Suppression of colorectal cancer liver metastasis by apolipoprotein(a) kringle V in a nude mouse model through the induction of apoptosis in tumor-associated endothelial cells.

Ahn JH, Yu HK, Lee HJ, Hong SW, Kim SJ, Kim JS - PLoS ONE (2014)

Bottom Line:
The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls.The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone.Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.

ABSTRACTThe formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.

pone-0093794-g004: Suppression of liver metastasis by rhLK8.Approximately 3×105 LS174T human colorectal carcinoma cells were injected into the spleen parenchyma of athymic BALB/c nude mice. Mice had daily i.p. administrations of rhLK8 (50, 10 or 2 mg/kg/day) for fourteen days. Mice were then sacrificed, and the livers were collected to analyze the metastasis of intrasplenically injected LS174T cells. (A) Representative photographs showing livers obtained from control (left) or rhLK8-treated (right) mice. (B) Number of surface tumor nodules in control and rhLK8-treated livers. *, p<0.05; **, p<0.01. (C) Sections of tumor tissues stained with H&E. Arrows indicate liver metastases. (D) The number of liver metastases per unit area (100 mm2) of randomly selected fields. *, p<0.05; **, p<0.01. (E) Immunohistochemical analyses of liver metastases of LS174T human colorectal carcinoma. Nuclei of liver specimens were stained with Hoechst 33342 (a and c), and double immunofluorescence staining was performed for CD31/PECAM-1 (red) and TUNEL (green) in control mice (a and b) or mice treated with rhLK8 (c, d, and e) as indicated. Apoptosis of tumor-associated endothelial cells (yellow) in the liver metastases treated with rhLK8 is indicated by arrows. Magnification, ×100. High magnification (panel e; ×200) of a selected region of panel (d), indicated by a dotted box. Bars, 100 μm. Data are representative of at least three independent experiments. (Replicates of Fig. 4A and 4B are available in Fig. S1).

Mentions:
As angiogenesis is critical for tumor cell metastasis and solid tumor growth, we examined the effects of rhLK8 on the liver metastasis of intrasplenically injected LS174T cells. Mice in the rhLK8 treatment group that received daily i.p. injections of 2, 10, or 50 mg/kg rhLK8 showed fewer liver metastases in a dose-dependent manner (Fig. 4A–B and Fig. S4) compared with the control group of mice. To further analyze rhLK8-mediated suppression of liver metastasis, the liver tissues were stained with H&E, and metastases were counted. There were significantly more metastasized tumors in the livers of control mice than in those of the rhLK8-treated mice (Fig. 4C). The number of liver metastases in rhLK8-treated mice was significantly lower than in control mice, and this effect was dose dependent (Fig. 4D).

pone-0093794-g004: Suppression of liver metastasis by rhLK8.Approximately 3×105 LS174T human colorectal carcinoma cells were injected into the spleen parenchyma of athymic BALB/c nude mice. Mice had daily i.p. administrations of rhLK8 (50, 10 or 2 mg/kg/day) for fourteen days. Mice were then sacrificed, and the livers were collected to analyze the metastasis of intrasplenically injected LS174T cells. (A) Representative photographs showing livers obtained from control (left) or rhLK8-treated (right) mice. (B) Number of surface tumor nodules in control and rhLK8-treated livers. *, p<0.05; **, p<0.01. (C) Sections of tumor tissues stained with H&E. Arrows indicate liver metastases. (D) The number of liver metastases per unit area (100 mm2) of randomly selected fields. *, p<0.05; **, p<0.01. (E) Immunohistochemical analyses of liver metastases of LS174T human colorectal carcinoma. Nuclei of liver specimens were stained with Hoechst 33342 (a and c), and double immunofluorescence staining was performed for CD31/PECAM-1 (red) and TUNEL (green) in control mice (a and b) or mice treated with rhLK8 (c, d, and e) as indicated. Apoptosis of tumor-associated endothelial cells (yellow) in the liver metastases treated with rhLK8 is indicated by arrows. Magnification, ×100. High magnification (panel e; ×200) of a selected region of panel (d), indicated by a dotted box. Bars, 100 μm. Data are representative of at least three independent experiments. (Replicates of Fig. 4A and 4B are available in Fig. S1).

Mentions:
As angiogenesis is critical for tumor cell metastasis and solid tumor growth, we examined the effects of rhLK8 on the liver metastasis of intrasplenically injected LS174T cells. Mice in the rhLK8 treatment group that received daily i.p. injections of 2, 10, or 50 mg/kg rhLK8 showed fewer liver metastases in a dose-dependent manner (Fig. 4A–B and Fig. S4) compared with the control group of mice. To further analyze rhLK8-mediated suppression of liver metastasis, the liver tissues were stained with H&E, and metastases were counted. There were significantly more metastasized tumors in the livers of control mice than in those of the rhLK8-treated mice (Fig. 4C). The number of liver metastases in rhLK8-treated mice was significantly lower than in control mice, and this effect was dose dependent (Fig. 4D).

Bottom Line:
The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls.The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone.Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.

ABSTRACTThe formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.